Conjugates for protection from nephrotoxic active substances

ABSTRACT

The present invention relates to a conjugate containing at least one kidney-selective carrier molecule and at least one active compound which has a protective action for the kidney against nephrotoxic active compounds, to a process for the preparation of the conjugate, to the use thereof for the protection of the kidney against nephrotoxic active compounds, and to a medicament comprising the conjugate.

The present invention relates to a conjugate containing at least onekidney-selective carrier molecule and at least one active compound whichhas a protective action for the kidney against nephrotoxic activecompounds, to a process for the preparation of the conjugate, to the usethereof for protection of the kidney against nephrotoxic activecompounds, and to a medicament comprising the conjugate.

The kidney is of importance, in particular, for the transport andexcretion of various substances and in the production of hormones. Onefunction of the kidneys is the excretion of end products of metabolism,the so-called uro-phanic substances, and toxins from the body throughthe formation of urine, which is finally excreted from the body via theurinary tract. The kidney regulates the water balance and thus servesfor long-term regulation of blood pressure. It regulates the electrolytebalance and the acid-base balance by control of the composition ofurine. Furthermore, the kidney is an important organ for intermediarymetabolism in the body (it effects gluconeogenesis). The kidney produceshormones, such as, for example, erythropoietin, for blood formation andis the site of degradation of peptide hormones. However, many functionsof the kidney itself are also controlled by hormones.

Today, about 280 million people suffer from chronic kidney diseases.Many diagnostic and therapeutic methods have already been developed. Forexample, immunosuppressants, cytostatics, immunotherapeutic agents,antiphlogistics, antibiotics, virostatics, antihypertensives,uricosurics, or diuretics are employed for the treatment of the kidneyor for influencing kidney function.

A number of approved active compounds, in particular cytostatics,exhibit nephrotoxicity as dose-limiting undesired side effect. Examplesof substances having a nephrotoxic action are cisplatin, carboplatin,gentamicin or cyclophosphamide. For example, the widespread cytostaticcisplatin damages the proximal tubule cells (PTCs) of the kidneys, sothat the dose in the case of single administration and the number oftherapy cycles are restricted. Damage to the PTCs is caused byintracellular oxidative stress (Matsushima H, et al. 1998, Journal ofLaboratory and Clinical Medicine, 131:518-526).

Amifostin is a cytoprotective medicament for which a chemo- andradioprotective action has been demonstrated in a large number of modelorganisms and in humans.

Amifostin itself is a prodrug which is cleaved by alkaline phosphataseslocated in the membrane of the endothelial cells to give the actualactive compound 2-((aminopropyl)amino)ethanethiol.

Alkaline phosphatases are expressed to a significantly lesser extent inmalignant tumour tissue than in healthy tissue. Consequently, amifostinis taken up principally by healthy cells. This selectivity—it is in theregion of 100:1—is necessary in order to avoid also developing thechemo- and radioprotective action in the tumour cells. The activespecies is the antioxidative thiol group of2-((aminopropyl)amino)ethanethiol.

Amifostin is not available in oral form. It is usually infused half anhour before radiotherapy or infusion of a chemotherapeutic agent. Thedose here is in the range from 740 to 900 mg/m² of body surface area. Inover half of patients, arterial hypotension is observed as a severe sideeffect.

There was therefore a need to protect the kidney better and morespecifically against nephrotoxic active compounds during treatment (forexample radiotherapy or infusion of a chemotherapeutic agent) in orderto prevent the side effects of the active compounds.

The object of the present invention was therefore the provision of asolution for protecting the kidney specifically against nephrotoxicactive compounds.

Surprisingly, it has been found that conjugates of kidney-selectivecarrier molecules and kidney-protecting active compounds are highlysuitable for achieving this object.

The present invention therefore relates to a conjugate containing atleast one kidney-selective carrier molecule and at least one activecompound which has a protective action for the kidney againstnephrotoxic active compounds.

In accordance with the invention, a kidney-selective carrier molecule istaken to mean a molecule which can serve as carrier (or transporter) foran active compound and enables targeted transport into the kidney. Acarrier molecule which can be used in accordance with the invention isany compound which has sufficiently high kidney selectivity afterconjugation with the active compound.

The prior art discloses, for example, the following substances which aresuitable for targeting of the kidney, i.e. for targeted transport intothe kidney:

Relatively small endogenous proteins, such as lysozyme (14.3 kDa), areable to pass through the glomerulus of the kidneys and are suitable astransporters for addressing of the kidneys with active compounds(Franssen et al.: J. Med. Chem. 35, 7, 1992, 1246-1259; Zhang et al.:Biomaterials 30, 2009, pp. 1372-1381).

Furthermore suitable in accordance with the invention are variouspeptides having about 5 to 20 amino acids which are taken up selectivelyby the kidneys. These are, for example, APASLYN and HITSLLS (Denby etal.: Molecular Therapy 15, 9, 2007, 1647-1654) or ANTPCGPYTHDCPVKR(Kumar and Deutscher: The Journal of Nuclear Medicine 49, 5, 2008,796-803; Geng et al.: Bioconjugate Chemistry 23, 2012, 1200-1210).

The kidney-selective carrier molecule is preferably a peptide whichcontains more than 50% (based on the number of amino acid units) ofsequence sections of the formula (1)-(A_(n)-B_(m)-C_(o))-  (1),whereA stands for an amino acid having an acidic side group,B stands for an amino group having a basic side group,C stands for any desired amino acid,n, m, independently of one another, stand for an integer from 1 to 10,where n:m=1:3 to 3:1,o stands for an integer between 0 and 10,and where

-   -   the peptide overall has a chain length of 5 to 100 amino acid        units    -   and the peptide consists of at least 50% (based on the number of        amino acid units) of amino acids A and B.

In accordance with the invention, a peptide is taken to mean a compoundwhich has formed from linking of two or more amino acids via amidebonds. The individual amino acids here are connected in a definedsequence to form a chain.

In accordance with the invention, amino acids are compounds which carryat least one amino group and at least one carboxyl group. Examples arenatural, proteinogenic amino acids or non-proteinogenic amino acidswhich occur in organisms or are prepared synthetically.

The amino acid units can be present in the D or L form in the peptide.

In accordance with the invention, the peptide comprises 5 to 100 aminoacids. In a preferred embodiment, the peptide has a chain length of 5 to40 amino acid units, particularly preferably a chain length of 10 to 30amino acid units.

In accordance with the invention, the peptide consists of more than 50%(based on the number of amino acid units) of sequence sections of theformula (1)-(A_(n)-B_(m)-C_(o))-  (1).

It preferably consists of more than 70% of sequence sections of theformula (1), particularly preferably more than 90%.

In formula (1), A stands for an amino acid having an acidic side group.This can be, for example, aspartic acid, glutamic acid,argininosuccinate and/or cysteic acid. Preference is given to aminoacids having a carboxyl function, i.e. glutamic acid and/or asparticacid, particularly preferably glutamic acid.

Within a peptide, A may stand for different amino acids having acidicside groups, i.e., for example, both glutamic acid and also asparticacid, argininosuccinate and/or cysteic acid residues may be presentsimultaneously in the peptide.

In an alternative embodiment, the amino acids having acidic side groupsA within a sequence section of the peptide are identical; in this case,for example, all amino acids A of the formula (1) in one sequencesection of the peptide stand for aspartic acid, glutamic acid,argininosuccinate or cysteic acid, and those in a further sequencesection of the peptide stand, independently of the above-mentionedsequence section, for aspartic acid, glutamic acid or cysteic acid.

In a further alternative embodiment, the amino acids having acidic sidegroups A within the peptide are identical; in this case, all amino acidsA of the peptide stand, for example, for aspartic acid, glutamic acid,argininosuccinate or cysteic acid.

In a preferred embodiment, all amino acids A within the peptide standfor glutamic acid.

n in formula (1) defines the number of amino acid units A. n here standsfor an integer from 1 to 10. n preferably stands for an integer from 1to 5, particularly preferably for 2 or 3.

In formula (1), B stands for an amino acid having a basic side group.This can be, for example, lysine, arginine, histidine and/or ornithine.Preference is given to lysine.

Within a peptide, B may stand for different amino acids having basicside groups, i.e., for example, both lysine, arginine, histidine and/orornithine residues may be present simultaneously in the peptide.

In an alternative embodiment, the amino acids having basic side groups Bwithin a sequence section of the peptide are identical; in this case,for example, all amino acids B of the formula (1) in one sequencesection of the peptide stand for lysine, arginine, histidine orornithine, and those in a further sequence section of the peptide stand,independently of the above-mentioned sequence section, for lysine,arginine, histidine or ornithine.

In a further alternative embodiment, the amino acids having basic sidegroups B within the peptide are identical; in this case, all amino acidsB of the peptide stand, for example, for lysine, arginine, histidine orornithine.

In a preferred embodiment, all amino acids B within the peptide standfor lysine.

m in formula (1) defines the number of amino acid units B. m here standsfor an integer from 1 to 10. m preferably stands for an integer from 1to 5, particularly preferably for 2 or 3.

In formula (1), C stands for any desired amino acid. This can be, forexample, alanine, arginine, asparagine, cysteine, glutamine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, valine and/orcitrulline.

Preference is given to proteinogenic amino acids which are linked in anatural manner. This ensures degradation of the peptide in the proximaltubule cells of the kidneys to give toxicologically entirely benignmetabolites.

Within a peptide, C may stand for different amino acids.

o in formula (1) defines the number of amino acid units C. o here standsfor an integer from 0 to 10. o preferably stands for 0, 1 or 2,particularly preferably for 0 or 1. In a very particularly preferredembodiment, o stands for 0, i.e. in this case no amino acid unit C ispresent in the peptide.

In a preferred embodiment, n and m stand, independently of one another,for 2 or 3.

In accordance with the invention, the ratio of n:m in formula (1) is 1:3to 3:1. Illustrative embodiments of the sequence sections of the formula(1) are: -(A₁-B₃-C_(o))-, -(A₁-B₂-C_(o))-, -(A₁-B₁-C_(o))-,-(A₂-B₆-C_(o))-, -(A₂-B₅-C_(o))-, -(A₂-B₄-C_(o))-, -(A₂-B₃-C_(o))-,-(A₂-B₂-C_(o))-, -(A₂-B₁-C_(o))-, -(A₃-B₉-C_(o))-, -(A₃-B₈-C_(o))-,-(A₃-B₇-C_(o))-, -(A₃-B₆-C_(o))-, -(A₃-B₅-C_(o))-, -(A₃-B₄-C_(o))-,-(A₃-B₃-C_(o))-, -(A₃-B₂-C_(o))-, -(A₃-B₁-C_(o))-, -(A₄-B₁₀-C_(o))-,-(A₄-B₉-C_(o))-, -(A₄-B₈-C_(o))-, -(A₄-B₇-C_(o))-, -(A₄-B₆-C_(o))-,-(A₄-B₅-C_(o))-, -(A₄-B₄-C_(o))-, -(A₄-B₃-C_(o))-, -(A₄-B₂-C_(o))-,-(A₅-B₁₀-C_(o))-, -(A₅-B₉-C_(o))-, -(A₅-B₈-C_(o))-, -(A₅-B₇-C_(o))-,-(A₅-B₆-C_(o))-, -(A₅-B₅-C_(o))-, -(A₅-B₄-C_(o))-, -(A₅-B₃-C_(o))-,-(A₅-B₂-C_(o))-, -(A₆-B₁₀-C_(o))-, -(A₆-B₉-C_(o))-, -(A₆-B₈-C_(o))-,-(A₆-B₇-C_(o))-, -(A₆-B₆-C_(o))-, -(A₆-B₅-C_(o))-, -(A₆-B₅-C_(o))-,-(A₆-B₃-C_(o))-, -(A₆-B₂-C_(o))-, -(A₇-B₁₀-C_(o))-, -(A₇-B₉-C_(o))-,-(A₇-B₈-C_(o))-, -(A₇-B₇-C_(o))-, -(A₇-B₆-C_(o))-, -(A₇-B₅-C_(o))-,-(A₇-B₄-C_(o))-, -(A₇-B₃-C_(o))-, -(A₈-B₁₀-C_(o))-, -(A₈-B₉-C_(o))-,-(A₈-B₈-C_(o))-, -(A₈-B₇-C_(o))-, -(A₈-B₈-C_(o))-, -(A₈-B₅-C_(o))-,-(A₈-B₄-C_(o))-, -(A₈-B₃-C_(o))-, -(A₉-B₁₀-C_(o))-, -(A₉-B₉-C_(o))-,-(A₉-B₈-C_(o))-, -(A₈-B₇-C_(o))-, -(A₉-B₆-C_(o))-, -(A₉-B₅-C_(o))-,-(A₉-B₄-C_(o))-, -(A₉-B₃-C_(o))-, -(A₁₀-B₁₀-C_(o))-, -(A₁₀-B₉-C_(o))-,-(A₁₀-B₈-C_(o))-, -(A₁₀-B₇-C_(o))-, -(A₁₀-B₆-C_(o))-, -(A₁₀-B₅-C_(o))-or -(A₁₀-B₄-C_(o))-, where A, B, C and o are defined as described above.

In accordance with the invention, the sequence of the formula (1) canstand, for example, for a sequence selected from.

-(EKKK)-, -(EKK)-, -(EK)-, -(EEKKKKK)-, -(EEKKKK)-, -(EEKKK)-, -(EEKK)-,-(EEK)-, -(EEEKKKKK)-, -(EEEKKKK)-, -(EEEKKK)-, -(EEEKK)-, -(EEEK)-,-(EEEEKKKKK)-, -(EEEEKKKK)-, -(EEEEKKK)-, -(EEEEKK)-, -(EEEEEKKKKK)-,-(EEEEEKKKK)-, -(EEEEEKKK)-, -(EEEEEEKK)-, -(DKKK)-, -(DKK)-, -(DK)-,-(DDKKKKK)-, -(DDKKKK)-, -(DDKKK)-, -(DDKK)-, -(DDK)-, -(DDDKKKKK)-,-(DDDKKKK)-, -(DDDKKK)-, -(DDDKK)-, -(DDDK)-, -(DDDDKKKKK)-,-(DDDDKKKK)-, -(DDDDKKK)-, -(DDDDKK)-, -(DDDDDKKKKK)-, -(DDDDDKKKK)-,-(DDDDDKKK)-, -(DDDDDDKK)-, -(ERRR)-, -(ERR)-, -(ER)-, -(EERRRRR)-,-(EERRRR)-, -(EERRR)-, -(EERR)-, -(EER)-, -(EEERRRRR)-, -(EEERRRR)-,-(EEERRR)-, -(EEERR)-, -(EEER)-, -(EEEERRRRR)-, -(EEEERRRR)-,-(EEEERRR)-, -(EEEERR)-, -(EEEEERRRRR)-, -(EEEEERRRR)-, -(EEEEERRR)-,-(EEEEEERR)-, -(EKRK)-, -(ERK)-, -(EDKKRRK)-, -(EDKKKK)-, -(ECKKH)-,-(EDKK)-, -(DEEKKKHK)-, -(EDDKKKK)-, -(EDERRR)-, -(DCEKH)-, -(DEEK)-,-(DEDERKRKR)-, -(DEEDKKKH)-, -(EDCEKRH)-, -(EDDEKK)-, -(EEEEEKKRRK)-,-(EEEEDKKRK)-, -(EDDEEKKR)-, -(DDEEEEKK)-,in each of which the one-letter codes of the amino acids are used: E(glutamic acid), D (aspartic acid), C (cysteine), K (lysine), R(arginine), H (histidine).

The sequence of the formula (1) preferably stands for a sequenceselected from the group comprising -(KKEEE)-, -(RREEE)-, -(KKEE)-,-(KKKEEE) and -(KKKEE)-.

The sequence of the formula (1) particularly preferably stands for thesequence -(KKEEE)-:

In accordance with the invention, the peptide consists of at least 50%(based on the number of amino acid units) of amino acids A and B. Thepeptide preferably consists of at least 70% (based on the number ofamino acid units) of amino acids A and B, particularly preferably atleast 80%.

In accordance with the invention, the sequence section of the formula(1) may be present in the peptide in total 1 to 50 times, preferably 1to 30 times, particularly preferably 1 to 10 times, especiallypreferably 2 to 5 times.

In a possible embodiment, the peptide contains a plurality of directlysuccessive sequence sections of the formula (1). The peptide preferablycontains 3 to 5 successive sequence sections of the formula (1).

For example, the peptide may consist of 3 to 5 successive sequencesections of the formula (1) and one or more further amino acids at the Cand/or N terminal. This is illustrated in formula (2):X_(p)(A_(n)B_(m)C_(o))_(x)Y_(q)  (2)in which A, B, C, n, m and o are as defined above,x stands for 3, 4, or 5,X and Y stand, independently of one another, for any desired amino acid,preferably for A, andp and q stand, independently of one another, for an integer between 0and 3, preferably for 0 or 1.

Examples of possible peptides in the conjugate according to theinvention are peptides selected from the group comprising (RREEE)₃R,(KKEE)₅K, (KKKEE)₃K, (KKKEEE)₃K and (KKEEE)₃K.

In an alternative embodiment of the present invention, thekidney-selective carrier molecule is an ε-polylysine conjugate, asdescribed in WO 2011/009539 A1. This carrier molecule likewise enableshighly selective concentration in the kidney. The lysine units in thepolymer are linked via their ε-amino groups.

The present invention therefore furthermore also relates to a conjugate,as described above, characterised in that the at least onekidney-selective carrier molecule is a conjugate (2), containing atleast one compound carrying carboxyl groups and an oligomer whichconsists of peptidically linked monomer units and which is either builtup from more than 50% (based on the number of monomer units) of lysinemonomer units, or contains at least 10 successive monomer units whichare built up from at least 70% (based on the number of monomer units) oflysine monomer units, where the above-mentioned lysine monomer units inthe oligomer are in each case linked via the ε-amino group of the sidechain,

characterised in that the proportion of carboxyl groups in the compoundcarrying carboxyl groups in the molecular weight of the compoundcarrying carboxyl groups is greater than 30%.

For the purposes of the present invention, the terms “ε-lysine monomerunits” and “ε-lysine units” used below stand for lysine monomer unitswhich are linked in the oligomer in each case via the ε-amino group oftheir side chain.

An ε-lysine unit has the following chemical structure:

The ε-lysine monomer units can be in the D or L form in the oligomer.

In a preferred embodiment, the oligomer has a chain length of 10 to 50monomer units.

In a preferred embodiment, at least one compound carrying carboxylgroups is bonded via the amino group of an ε-lysine monomer unit, i.e.one or more ε-lysine monomer units carry on their amino group a compoundcarrying carboxyl groups which is conjugated directly or via a spacer.

In an embodiment, the compound carrying carboxyl groups is a complexingagent, particularly preferably DOTA (=1,4,7,10-tetraazacyclododecane-N,-N′, -N″, -N′″-tetraacetic acid) or DTPA (diethylenetriaminepentaaceticacid).

A compound carrying carboxyl groups is a chemical compound whichcontains at least one carboxyl group (—COOH) and at least one group orfunctionality for bonding to the oligomer of conjugate (2). The bondingto the oligomer can take place in any known manner which results incovalent bonding of the oligomer and compound carrying carboxyl groups.Examples of functional groups via which bonding can take place are —NH₂,—SH, —OH, -Hal (for example —Cl, —Br, —I), -alkyne, —NCS, —NCO, —SO₂Cl,-azide, -carbonate, -aldehyde, -epoxide, —COOH, —COOR, where R in thiscase is preferably a halogen or preferably an activator, i.e. a goodleaving group, such as, for example, N-hydroxysuccinimide,pentafluorophenyl or para-nitrophenyl. An overview of possible covalenttypes of coupling is found, for example, in “Bioconjugate Techniques”,Greg T. Hermanson, Academic Press, 1996, on pages 137 to 165.

The compound carrying carboxyl groups preferably contains two or morecarboxyl groups. These can be bonded directly or via a spacer to thecarboxyl- and/or amino-terminal end of the oligomer and/or to functionalgroup of the monomer units which is suitable for conjugation (forexample NH, —NH₂, —COOH, —OH, —SH, -Hal (for example —Cl, —Br, —I),-alkyne, -azide, -aldehyde). Examples of compounds carrying carboxylgroups which are suitable in accordance with the invention are: citricacid, succinic acid, fumaric acid, maleic acid, glutamic acid, adipicacid, tartaric acid, oxalic acid, malonic acid, glutaric acid, adipicacid, suberic acid, azelaic acid, sebacic acid, the correspondingbranched fatty acids, maleic acid, fumaric acid, cyclohexanedicarboxylicacid and the corresponding position isomers and similar aliphaticdibasic acids; tetrahydrophthalic acid, 5-norbornene-2,3-dicarboxylicacid and similar alicyclic dibasic acids; tricarballylic acid, aconiticacid, trimesic acid and similar tribasic acids;adamantanetetracarboxylic acid, butanetetracarboxylic acid,cyclopentanetetracarboxylic acid, tetrahydrofurantetracarboxylic acidand similar tetrabasic acids; sugar acids, in particular aldaric acids,such as, for example, glucaric acid, galactaric acid; malic acid,tartaric acid, citric acid and similar hydroxyfatty acids; trimelliticacid, pyromellitic acid, biphenyltetracarboxylic acid,benzophenonetetracarboxylic acid, diphenylsulfonetetracarboxylic acidand similar aromatic polycarboxylic acids.

In accordance with the invention, the compound carrying carboxyl groupscan also be complexing agents which contain at least one carboxyl group,preferably two or more carboxyl groups, and at least one group orfunctionality for bonding to the oligomer of the conjugate according tothe invention. Examples thereof are NOTA, TETA, EDTA or preferably DOTAor DTPA.

The compounds carrying carboxyl groups are typically bound via aminogroups of the monomer units, for example the free amino group ofε-lysine.

Preferred compounds carrying carboxyl groups are those which contain twoor more free carboxyl groups after conjugation to the oligomer.

It has been found that the specificity achieved in the targeting of thekidney is particularly high if the carboxyl groups of the compoundcarrying carboxyl groups make up a large proportion of the molar mass ofthe compound carrying carboxyl groups. Preference is therefore given tocompounds carrying carboxyl groups in which the proportion of thecarboxyl groups in the molar mass is greater than 30%, preferablygreater than 40%.

Compounds carrying carboxyl groups which are particularly preferred inaccordance with the invention are therefore those which contain two ormore free carboxyl groups after conjugation to the oligomer and in whichthe proportion of the carboxyl groups in the molar mass is greater than30%, preferably greater than 40%, such as, for example, DOTA, DTPA andcitric acid.

It has been found that the conjugates accumulate particularlyspecifically in the kidney if a compound carrying carboxyl groups iscovalently bonded to 10 to 80% of the monomer units.

The conjugate (2) should preferably contain at least one compoundcarrying carboxyl groups per 10 monomer units, particularly preferablybetween 3 and 6 compounds carrying carboxyl groups per 10 monomer units.Equally, however, it is also possible for one compound carrying carboxylgroups to be bonded to more than 9 of 10 monomer units or to all monomerunits. The optimum number of compounds carrying carboxyl groups per 10monomer units depends on the type of the compound carrying carboxylgroups and the type of the monomer units. The above-mentioned preferrednumber of compounds carrying carboxyl groups per monomer unit apply, inparticular, to oligomers which are built up entirely from ε-lysinemonomer units. The distribution of the compounds carrying carboxylgroups in conjugate (2) can be random, meaning that, for example, thefirst monomer units contain —NH₂, followed by a monomer unit with acompound carrying carboxyl groups, then again one containing —NH₂, thentwo times a monomer unit which carries a compound carrying carboxylgroups, then again twice one containing —NH₂, etc.

The term oligomer denotes the part of conjugate (2) that consists of anoligomer which consists of peptidically linked monomer units. Theoligomer typically consists of 5 to 1000, preferably 8 to 100,particularly preferably 10 to 50, monomer units. In a particularlypreferred embodiment, the oligomer consists of ε-polylysine which hasbetween 8 and 100 monomer units, particularly preferably between 10 and50 monomer units.

In other embodiments, however, up to 50% of the ε-lysine monomer unitsmay be replaced by other monomer units and/or up to 50% of the ε-lysinemonomer units may be derivatised or modified by the introduction offurther functionalities. Likewise, the oligomer which consists ofpeptidically linked monomer units may contain a plurality of successivemonomer units which are not ε-lysine monomer units if it contains atleast 10 successive monomer units which consist of at least 70% (basedon the number of monomer units), preferably at least 80%, of ε-lysineunits. This is the case, for example, if a chain of 10 to 20 monomerunits (for example comprising amino acids) in which no ε-lysine monomerunit is present and subsequently, for example, ten monomer units, ofwhich eight are ε-lysine monomer units and two consist of other aminoacids, is located at one end of the oligomer.

In accordance with the invention, the term monomer unit denotes any partof the oligomer that is peptidically linked to at least one further partof the oligomer. Terminal monomer units here are generally onlypeptidically linked to one further monomer unit. Monomer units in themiddle of the oligomer are peptidically linked to two further monomerunits. Monomer units which are peptidically linked to three furthermonomers are located at branching points. In the case of monomer unitsin the middle of the oligomer, the monomer unit typically provides onthe one hand the —NH part of the peptidic bond and on the other hand the—CO part.

Typical other monomer units which the oligomer according to theinvention can contain besides the ε-lysine monomer units are natural orsynthetic amino acids, such as, in particular, alanine, β-alanine,glycine, glutamic acid, aspartic acid or arginine.

Further typical monomer units are monomer units having a spacer functionof the formula—NH—SP—CO—  Iwhere SP can be a C1 to C20 alkylene, alkenylene or alkynylene group, inwhich one or more non-adjacent methylene groups may be replaced by —O—,—S—, —S(O)—, —SO₂—, —SO₂O—, —C(O)—, —C(O)O—, —CH₂—, —CHR′—, —CR′₂—,—CR′═CH—, —CH—CR′—, —CH═CH—, —CR′═CR′—, —C≡C—, —N⁺R′₂—, —P(O)R′O—,—C(O)NR′—, —SO₂NR′—, —OP(O)R′O—, —P(O)(NR′₂)NR′—, —PR′₂═N- or —P(O)R′—where R′═C₁- to C₆-alkyl, C₃- to C₇-cycloalkyl, unsubstituted orsubstituted phenyl.

SP preferably stands for linear C3 to C10-alkyl chains, linear C3-C10chains having one or more alkylene groups, for ethylene glycol chainshaving two to ten ethylene glycol units or for oligopeptide chains.

Further typical monomer units are those which contain functionalitiesfor the linking of spacers, active compounds, peptides, dyes,solubilisers, protecting groups, a solid phase or similar components orto which components such as active compounds, complexing agents,peptides, solubilisers, protecting groups, a solid phase or dyes arealready bonded directly or via a spacer. Monomer units of this typepreferably have at least one of the following functional groups —NH₂,—SH, —OH, -Hal (for example —Cl, —Br, —I), -alkyne, —NCS, —NCO, —SO₂Cl,-azide, -carbonate, -aldehyde, -epoxide, —COOH, —COOR, where R in thiscase is preferably a halogen or preferably an activator, i.e. a goodleaving group, such as, for example, N-hydroxysuccinimide,pentafluorophenyl or para-nitrophenyl, or are linked to activecompounds, complexing agents, peptides, dyes or similar components via afunctional group of this type.

Furthermore, the oligomer according to the invention may containε-lysine monomer units which are derivatised. These are monomer units inwhich further functionalities (F1/F2) are correspondingly bonded to theNH group and/or the amino group.

In a preferred embodiment, the oligomer in conjugate (2) contains theamino acid cysteine. This embodiment has the advantage that the activecompound can be bonded directly to the SH group of cysteine forprotection of the kidneys. This bond can easily be brokenintracellularly, enabling on the one hand the active compound to bereleased and on the other hand the SH group on the cysteine residue,which may itself have an antioxidative action, to become free again.

It is obvious to the person skilled in the art that the formulaedepicted above depict monomer units in the middle of the oligomer chainand that terminal monomer units, depending on whether they are locatedat the C- or N-terminal end, in each case carry a COOH or COOR groupinstead of —CO— or carry an NH₂, NF1H, NF1R, NHR or NR₂ group instead of—NH— or —NF1-, where R is typically H, linear or branched C1-C6 alkyl, aspacer function for the bonding of active compounds, complexing agents,peptides, dyes, solubilisers, protecting groups, a solid phase orsimilar components, or an active compound, complexing agent, peptide,dye, solubiliser, protecting group, a solid phase or similar componentbonded directly or via a spacer.

In accordance with the invention, an active compound which has aprotective action for the kidney against nephrotoxic active compounds istaken to mean an active compound which reduces damage to the proximaltubule cells. For example, this is an active compound selected fromantioxidants, apoptosis inhibitors, active compounds having an influenceon the cell cycle, active compounds which activate the repair mechanismsof the cells, and combinations thereof.

In principle, damage to the proximal tubule cells can be reduced onseveral levels with the aid of these active compounds:

In the first step, the uptake of cytotoxic compounds into the interiorof the cells can be prevented by blockade of the transport mechanisms ofthe proximal tubule cells. The blockade can take place through specificinhibitors or alternatively also through sufficient amounts of thetransport molecule itself, which temporarily blocks the receptors of theproximal tubule cells. A similar action is exhibited by substances whichreduce the metabolic activity of the proximal tubule cells, or allowthese cells to remain in the G₀ phase of the cell cycle.

In the second step, nephrotoxic compounds which have been transported ordiffused into the cell can be rendered harmless by “antidotes” whichhave been channeled into the cell interior via the active compoundtransporter before administration of the nephrotoxic active compound.

The aim of the third step is to suppress apoptosis of damaged proximaltubule cells. Cytotoxic substances, such as, for example, cisplatin,cause damage to the DNA in the cell nucleus. If the damage level exceedsa certain threshold, programmed cell death, apoptosis, is triggered inthe cell. In the case of tumour cells, this process is desired, but isfatal in the case of the proximal tumour cells of the kidneys. Somesubstances (apoptosis inhibitors) are known which are capable ofpreventing programmed cell death, or increasing the threshold for theinitiation of apoptosis in the cell. In the fourth step, substanceswhich activate the natural repair mechanisms of the cells and thusrepair the DNA damage can be channeled into the cells.

Blockade of the Transport Mechanisms (Step 1):

Active compounds which have an influence on the transport mechanisms ofthe proximal tubule cells can be conjugated onto the kidney-selectivecarrier molecules. Cellular transporters both on the apical side of theproximal tubule cells, that is the side which is in the lumen and is incontact with the glomerular ultrafiltrate, and also on the basolateralside, that is the side which faces the blood vessels, can be blockedthrough specifically selected active compound molecules. Transporters ofthe basolateral side of the proximal tubule cells are of majorimportance for proximal tubule secretion of endogenous substances andforeign substances, such as, for example, medicaments. Anionicsubstances are taken up by the proximal tubule cells via organic aniontransporter 1 (OAT1). Cationic substances, by contrast, via organiccation transporter 2 (OCT2). Both transporters can be inhibited bycertain active compounds. An example of an inhibitor of OAT1 is the drugprobenecid (Kurtz A, et al. 2009, Physiologie, ISBN 3-131-51496-5, p.365). Probenecid can be conjugated onto a peptide according to theinvention and, after glomerular filtration, taken up via the apical sideof the proximal tubule cells. Through a labile linker, for example anester group, by means of which the carboxyl group of probenecid isconjugated with the carrier molecule, the chemically unchanged activecompound in the endosome of the proximal tubule cells can be liberatedby esterases. The free active compound can reach the basolateral sidethrough diffusion or transporters and block the organic aniontransporter 1 there.

An example of an inhibitor of the organic cation transporter 2 is thedrug tacrine (Sung J H, et al. 2005, Drug Metab Dispos, 33(3):440-448PMID 15547049). Tacrine can also conjugate to a peptide according to theinvention, for example as Schiff's base or amidically. The organiccation transporter 2 can thus be blocked by the route described in theexample of conjugated probenecid. For example, the kidney-toxiccytostatic cisplatin is taken up and accumulated by the proximal tubulecells via OCT2 on the basolateral side. The cisplatin then develops itskidney-damaging action in the proximal tubule cells (Freissmuth M, etal. 2012, Pharmakologie & Toxikologie, ISBN 3-642-12353-8, p. 735).

Antioxidants (Step 2):

A number of substances with an antioxidative action can be conjugatedonto the kidney-selective carrier molecules. Suitable classes of activecompound are, inter alia, polyphenols (resveratrol, caffeic acid,luteolin, quercetin, rutin, cyanidin, xanthohumol, . . . ), lipoic acid,ascorbic acid, nicotinic acid, amifostin, alliin, thiols (for example2-mercaptoethanesulfonate-sodium (mesna)), tocopherols, carotinoidsand/or butylhydroxytoluene (BHT), or combinations thereof.

If the kidney-selective carrier molecule used is an ε-polylysineconjugate, as disclosed in WO 2011/009539 A1, the molecular structure ofthe oligomer can be varied by incorporating building blocks of the aminoacid cysteine into the oligomer. Cysteine has a free thiol group havingan antioxidative action. With this structure, the carrier can bemodified to give an active medicament with an antioxidative action.Independently thereof, a number of protective active compounds can beconjugated onto the peptidic carrier instead or in addition, asdescribed above.

Apoptosis Inhibitors (Step 3):

Anti-apoptotic substances can be conjugated onto the renal activecompound transporter. Examples of this group of active compounds arepifithrin-μ (Nijboer et al. 2011, Ann Neurol.:doi: 10.100²/ana.22413)and pifithrin-α (Komarova et al. 2000, Biochemistry (Mosc) 65(1):41-48)as well as MDL 28170 (Kawamura et al. 2005, Brain Res. 1037(1-2):59-69)and NS3694 (Zhao et al. 2010, Age (Dordr). 32(2):161-177). Pifithrin-α,a p53 inhibitor, is able considerably to raise the threshold for thetriggering of apoptosis in treated cells and model organisms.

An advantage of apoptosis inhibitors is that they can also beadministered after damage to the cells. The disadvantage of systemicadministration of apoptosis inhibitors, which may have the consequenceof an increased risk of cancer, can advantageously be prevented byorgan-specific administration—with the carrier molecule according to theinvention.

Active Compounds Having an Influence on the Cell Cycle or Metabolism:

Besides the antioxidants and the apoptosis inhibitors, compounds whichcause (temporary) stoppage of the cell cycle of the proximal tubulecells are likewise potential active compounds with which the damage tothe kidneys by nephrotoxic medicaments can be reduced. An examplethereof is the compound apigenin (Ruela-de-Sousa et al. 2010, Cell death& disease. 1, e19).

Active Compounds which Activate the Repair Mechanisms of the Cells (Step4):

By activation of certain transcription factors, such as, for example,Sp1, or MDC1 (Luo et al. 2012, The EMBO journal, 31(13):3008-3019), acell can be stimulated to increased repair of (double) strand breakages(Beishline et al. 2012, Molecular and cellular biology, DOI:10.1128/MCB.00049-12. PMID 22826432). The flavonoid baicalein(5,6,7-trihydroxyflavone) is an example of a compound which is able toactivate DNA repair in cells (Kim et al. 2012, Cell Biol Toxicol, DOI:10.1007/s10565-012-9233-y. PMID 23011636).

In addition, cell cycle arrest (Step 1) puts the cell in a state inwhich essentially “repair work” is carried out on the damaged DNA (Salehet al. 2012, Cancer biology & therapy 11, PMID 22895066).

The (simultaneous) administration of a number of classes of activecompound (Steps 1 to 4 of the preceding examples) is particularlyadvantageous for keeping damage to the proximal tubule cells as low aspossible. The individual active compounds here can either be conjugatedonto separate transporter molecules or a plurality of different activecompounds can be conjugated onto one transporter molecule.

Possible active compounds in the conjugate can therefore be selectedfrom the group comprising antioxidants, apoptosis inhibitors, activecompounds having an influence on the cell cycle, active compounds whichactivate the repair mechanisms of the cells, receptor inhibitors andcombinations thereof.

In a preferred embodiment, the active compound which has a protectiveaction for the kidney against nephrotoxic active compounds is anantioxidant and/or an apoptosis inhibitor.

Preferred antioxidants are lipoic acid, resveratrol, caffeic acid,luteolin, quercetin, rutin, cyanidin, xanthohumol, ascorbic acid,nicotinic acid, amifostin, alliin, thiols, mesna, tocopherols,carotinoids and butylhydroxytoluene (BHT).

Preferred apoptosis inhibitors are pifithrin-μ (Nijboer et al. 2011, AnnNeurol.:doi: 10.1002/ana.22413), pifithrin-α (Komarova et al. 2000,Biochemistry (Mosc) 65(1):41-48), MDL 28170 (Kawamura et al. 2005, BrainRes. 1037(1-2):59-69) and NS3694 (Zhao et al. 2010, Age (Dordr).32(2):161-177).

In a particularly preferred embodiment of the present invention, theactive compound which has a protective action for the kidney againstnephrotoxic active compounds is therefore resveratrol, caffeic acid,luteolin, quercetin, rutin, cyanidin, xanthohumol, ascorbic acid,nicotinic acid, amifostin, alliin, thiols, tocopherols, carotinoids,butylhydroxytoluene (BHT), pifithrin-μ, pifithrin-α, MDL 28170 and/orNS3694, or mixtures thereof.

In accordance with the invention, the conjugate contains at least onekidney-selective carrier molecule, as defined above, and at least oneactive compound which has a protective action for the kidney againstnephrotoxic active compounds, as defined above.

The bonding of the active compound to the carrier molecule is preferablycovalent and can optionally take place via a spacer.

In accordance with the invention, one or more identical or differentactive compound molecules may be bonded per conjugate according to theinvention.

Equally, the conjugate according to the invention, in particular in thecase of macromolecules, such as relatively large active compoundmolecules, for example proteins, may also contain two or more carriermolecules which are bonded to one active compound molecule in order tofacilitate kidney-specific concentration of the active compound. Thecarrier molecules are typically again covalently bonded to themacromolecule here. In accordance with the invention, macromolecules aretaken to mean not only large molecules such as proteins, but insteadalso any form of particles (for example nanoparticles), liposomes orother systems by means of which active compounds can be transported orbonded to the active compounds.

Furthermore, functionalities for cell-specific targeting, such as, forexample, antibodies, antibody fragments or aptamers, may be bonded tothe conjugate according to the invention. Fluorescent dyes orinterleukins, such as IL-2, may also be bonded.

The active compounds or other functionalities can be covalently bondedto the peptide directly or by means of a spacer.

A spacer, often also called linker, effects a covalent bond between twoparts of a molecule, in the present case, for example, between thepeptide and an active compound. A spacer is introduced, for example, ifthe connection between two moieties is not to take place only via adirect chemical bond, but instead a certain separation is to begenerated between two moieties. Equally, a spacer can provide thechemical functionalities which are necessary in order to connect twoparts of a molecule which would otherwise not react with one another.The conjugation of a spacer onto the carrier molecule or an activecompound preferably takes place via an amide or ester bond. Spacers canbe, for example, aliphatic hydrocarbons, polyethers (such aspolyethylene glycols), peptides or similar elements having a chainstructure. The spacer may be stable, i.e. it can only be cleaved to aslight extent or not at all under physiological conditions, or it may beunstable, i.e. it can be cleaved at least under certain physiologicalconditions.

Examples of functional groups via which direct bonding can take placeare —NH₂, —SH, —OH, -Hal (e.g. —Cl, —Br, —I), -alkyne, —NCS, —NCO,SO₂Cl, -azide, -carbonate, -aldehyde, -epoxide, —COOH, —COOR, where R inthis case is preferably a halogen or preferably an activator, i.e. agood leaving group, for example N-hydroxysuccinimide, pentafluorophenylor para-nitrophenyl. An overview of possible covalent types of couplingcan be found, for example, in “Bioconjugate Techniques”, Greg T.Hermanson, Academic Press, 1996 on pages 137 to 165.

For example, active compounds may be bonded via a cleavable linker inthe conjugate according to the invention. This linker is then cleaved invivo under certain conditions, for example enzymatically or chemically,and releases the active compound. For this purpose, suitable linkers arethose which contain carboxylate and disulfide bonds, in which the formergroups are hydrolysed enzymatically or chemically and the latter areseparated off by disulfide exchange, for example in the presence ofglutathione.

An example of a cleavable spacer is also a peptide which can be cleavedspecifically with the aid of specific, endogenous enzymes oralternatively those which are added to the body. Thus, for example, thepeptide sequence DEVD (Asp-Glu-Val-Asp) is cleaved after apoptosisinduction by caspase-3. For example, an active compound which is bondedvia a spacer of this type can thus be removed from the kidney after acertain residence time therein, or alternatively a correspondingfunctionality (presence or absence of a certain enzyme) of the kidneycan be checked. Further examples are the peptide sequences CPEN↓FFWGGGG(Salinas et al. 2008, Biomaterials 29, 2370-2377) or PENFF, which can becleaved by the matrix metalloprotease-13.

A simple embodiment of a cleavable spacer is the formation of acarboxylate, which can easily be cleaved by esterases.

In a preferred embodiment of the present invention, the active compoundis therefore bonded via an ester link. This enables precise cleaving-offof the active compound molecule in the kidney. At the same time,however, the link is previously sufficiently stable for transport intothe kidney in order to prevent premature cleaving-off.

Furthermore, a readily cleavable ester link of the active compound tothe active compound transporter enables relatively fast release of theactive compound at the target site. The cleavage of the ester link takesplace more quickly in terms of time than the degradation of the activecompound transporter by proteases.

Alternatively, the spacer may contain an acid-labile structure, forexample a hydrazone, an imine, a carboxylhydrazone, an acetal or ketal(see, for example, Haag-ft Kratz-F, Angewandte Chemie page 1218 (2006)).

In accordance with the invention, the at least one active compound canbe bonded to the N and/or C terminal of the carrier molecule.

In an alternative embodiment, the active compound can be bonded to anamino acid in the chain.

In a further alternative embodiment, the active compound can be bondedin the chain between the amino acids.

The conjugate according to the invention is taken up highly selectivelyby the kidneys and broken down relatively rapidly.

A suitable choice of the linking site of the active compound on thecarrier molecule, and a suitable choice of the chain length andmolecular structure of the carrier molecule, enables the desiredpharmacokinetics, i.e. the desired active compound release at the targetsite, i.e. in the kidney, to be established here.

Typically, longer carrier molecules result in delayed release comparedwith shorter carrier molecules. Longer carrier molecules have, forexample, chain lengths of 20 to 40 amino acids, preferably 30 aminoacids, while shorter carrier molecules are typically taken to mean chainlengths of 3 to 10 amino acids, preferably 5 amino acids.

The release of active compounds linked at the C terminal takes placesignificantly more quickly than that of active compounds linked at the Nterminal. Without being tied to this theory, it is assumed that therate-determining step in peptide degradation is influenced, inparticular, by carboxypeptidases, which break down the peptide startingfrom the C terminal.

In accordance with the invention, active compounds incorporated into thechain in a branched manner are also released significantly more slowlythan those linked in a linear manner. The enzymatic degradation ofbranched peptide structures is basically significantly more difficultthan the degradation of linear peptides.

Furthermore, the release rate of the active compound can, in accordancewith the invention, also be controlled by the type of linking thereof tothe oligomer. A readily cleavable ester link enables relatively fastrelease of the active compound at the target site (see above).

The present invention also relates to a process for the preparation of aconjugate, as described above, characterised in that an optionallyactivated active compound which has a protective action for the kidneyagainst nephrotoxic substances is conjugated onto the carrier molecule.

The preparation of the conjugates according to the invention typicallyhas at least the following process steps:

-   a) provision of a carrier molecule, as defined above, which contains    at least one reactive group,-   b) conjugation of at least one optionally activated active compound,    as defined above, to the carrier molecule from step a).

The carrier molecules of the conjugates according to the invention canbe prepared, in particular, by various processes known to the personskilled in the art in the area of peptide synthesis.

The preparation is typically carried out via a solid-phase synthesis.

In accordance with the invention, a solid phase is an organic, inorganicor organic/inorganic composite material which can be employed as resinor support in solid-phase synthesis. Furthermore, surfaces of mouldings,such as, for example, microtitre plates or particulate materials, suchas, for example, organic or inorganic nanoparticles, metal particles orthe like, are also regarded as solid phase in accordance with theinvention.

The solid-phase synthesis is carried out in a corresponding manner to aconventional peptide synthesis (for example Fmoc/tBu peptide synthesisor Boc/benzyl peptide synthesis). Solid-phase syntheses of this type areknown to the person skilled in the art. Suitable textbooks for peptidesynthesis are “Solid-Phase Peptide Synthesis”: 289 (Methods inEnzymology) by Sidney P. Colowick (author), Gregg B. Fields (publisher),Melvin I. Simon (publisher) Academic Press Inc (November 1997) or “FmocSolid Phase Peptide Synthesis: A Practical Approach” by W. Chan(author), W. C. Chan (publisher), Peter D. White (publisher) “OxfordUniv Pr (2 Mar. 2000). The monomers employed in each case are selectedhere in such a way that a peptide corresponding to the present inventionis formed. Depending on the type of amino acid unit, the synthesis canbe carried out using a derivatised amino acid unit directly or an aminoacid unit which is firstly protected at the site intended for thederivatisation. When the synthesis of the peptide is complete, the finalderivatisation with the active compound can then be carried out eitherin the solid phase or in solution after cleaving-off from the solidphase.

The bonding of the active compound in this case preferably takes placeto the finished peptide, i.e. either still on the solid phase when thesolid-phase synthesis of the peptide is complete or after the latter hasbeen cleaved off in solution.

If the active compound is to be bonded, for example, to the N-terminalend of the peptide, the peptides are typically generated with anamino-terminal protecting group, such as, for example, Fmoc. If theactive compound is able to withstand the conditions used on the one handfor cleaving off the peptide from the synthesis resin and on the otherhand for deprotecting the side chains, the Fmoc group can be cleaved offfrom the N terminal of the complete resin-bonded peptide, enabling theactive compound to be bonded to the free N-terminal amine. In suchcases, the active compound is typically activated by processes which aregenerally known in the art for producing an active ester or activecarbonate group which is effective for forming an amide or carbamatebond to the oligomer amino group. It is of course also possible to use adifferent linking chemistry.

In order to minimise side reactions here, guanidino and amidino groupsmay be blocked using conventional protecting groups, such as, forexample, carbobenzyloxy groups (CBZ), di-t-BOC, PMC, Pbf, N—NO₂ and thelike.

Coupling reactions are carried out by known coupling processes insolvents, such as, for example, N,N-dimethylformamide (DMF),N-methylpyrrolidone (NMP), dichloromethane (DCM) and/or water.Illustrative coupling reagents includeO-benzotriazolyloxytetramethyluronium hexafluorophosphate (HATU),dicyclohexylcarbodiimide, bromo-tris(pyrrolidino)phosphonium bromide(PyBroP), etc. Other reagents may be present, such as, for example,N,N-dimethylaminopyridine (DMAP), 4-pyrrolidinopyridine,N-hydroxysuccinimide or N-hydroxybenzotriazole (HOBt).

A carrier molecule based on an ε-polylysine conjugate, as described inWO 2011/009539 A1, can also be prepared starting from ε-polylysine.Typically, synthetic or natural ε-polylysine of uniform or differentchain length is reacted here in solution with the correspondingcompounds carrying carboxyl groups. To this end, for example, firstlythe compounds carrying carboxyl groups can be activated. This can becarried out, for example, by activation of one or more of their carboxylgroups by converting them into the active ester or acid chloride. Thisis followed by the reaction with ε-polylysine, with the conjugationpreferably taking place onto the free amino groups. Alternatively, forexample, one or more carboxyl groups of the compound carrying carboxylgroups can be activated by means of a coupling reagent, such asdicyclohexylcarbodiimide (DCC) or HATU, and reacted with theε-polylysine, with the conjugation preferably taking place onto the freeamino groups. Reaction conditions for reactions of this type are knownto the person skilled in the art. Suitable solvents are, for example,water, acetonitrile, DMSO, DMF, dioxane, THF, methanol or mixtures oftwo or more of the said solvents.

The conjugates according to the invention have the advantage thatsystemic side effects of active compounds for the treatment or imagingof the kidney can be substantially suppressed, since the conjugatesenable targeted transport of kidney-protecting substances into thekidney. These kidney-damaging active compounds include, for example,cisplatin, carboplatin, gentamicin and cyclophosphamide. In the case ofthese substances, the nephrotoxicity limits the dose or the number oftherapy cycles.

In connection with the treatment of the kidney with kidney-damagingsubstances, the administration of the conjugates according to theinvention (for example by injection into the bloodstream or aftersubcutaneous injection) before, during or after the therapy enablestargeted concentration of the protecting active compounds in the kidney.

The patient treated with a kidney-damaging substance, for examplecisplatin, is given an injection of the kidney-protecting conjugateabout 30 minutes before commencement of the therapy. Thekidney-protecting conjugate can take place here by a single injection orby continuous infusion over the entire period of cisplatinadministration, which usually extends over a period of one to two hours.

The present invention therefore also relates to a conjugate according tothe invention, as described above, as medicament, such as, inparticular, a therapeutic composition, in particular for protection ofthe kidney against nephrotoxic active compounds.

The present invention also relates to the use of a conjugate accordingto the invention, as described above, for protection of the kidneyagainst nephrotoxic active compounds.

In this connection, the use of a carrier molecule of the formula (1), asdefined above, is particularly advantageous, since this is extremelywell suited to targeting of the kidney: compared with other knownlow-molecular-weight structures, it also exhibits very goodconcentration in the kidney in conjugation with the active compound. Thecomparison with peptides described in the literature which are taken upselectively by the kidneys (APASLYN and HITSLLS, amino acids areindicated in single-letter code (Denby et al.: Molecular Therapy 15, 9,2007, 1647-1654)) shows that, although most peptides have more or lesshighly pronounced kidney selectivity after intravenous administration,this is not the case in conjugation with an active compound. However,the pharmacological usefulness of the peptide structures as transportsystem for targeted protection of the kidney against nephrotoxicsubstances only arises if these peptides are taken up together withconjugated active compounds virtually exclusively by the kidneys, namelythe proximal tubule cells. Only in this case does a significantadvantage arise over systemic administration of the active compound.

Furthermore, the conjugates according to the invention enablesubcutaneous and intraperitoneal administration of the peptide/activecompound conjugates according to the invention to successfully addressthe kidneys besides the intravenous administration of peptides/proteinsdescribed in the literature for active compound transport into thekidneys.

The intraperitoneal, and specifically the subcutaneous administrationroute is advantageous for the administration of a potential activecompound, compared with the intravenous route, for doctor and patient.

The present invention also relates to a medicament or a pharmaceuticalcomposition, in particular a therapeutic or image-enhancing composition,comprising at least one conjugate according to the invention, asdescribed above.

In accordance with the invention, the conjugate may also be in the formof its pharmaceutically usable salts and stereoisomers, includingmixtures thereof in all ratios.

The use of the conjugates according to the invention for the preparationof a pharmaceutical composition or a medicament, in particular atherapeutic composition, is also in accordance with the invention.

In accordance with the invention, the present invention can also relateto a kit for the preparation of a medicament or a pharmaceuticalcomposition, in particular a therapeutic composition, comprising atleast one conjugate according to the invention. This conjugate can thenbe reacted, for example, with a suitable active compound, depending onthe application, for the preparation of a therapeutic composition.

The present invention additionally relates to the conjugates accordingto the invention, and/or pharmaceutically usable salts and stereoisomersthereof, including mixtures thereof in all ratios, and optionallyexcipients and/or adjuvants

-   -   as medicament    -   for use as medicament    -   as active compound or active component in a medicament    -   for use for protection of the kidney against nephrotoxic active        compounds    -   and in particular as medicament for the treatment of diseases of        the kidney.

A therapeutic composition, a pharmaceutical composition or a medicamentgenerally consists at least of the active compound—in this case theconjugate according to the invention with bonded active compound—and oneor more suitable solvents and/or excipients which allow application ofthe therapeutic composition.

Pharmaceutical compositions or medicaments can be adapted foradministration via any desired suitable method, for example by oral(including buccal or sublingual), rectal, nasal, topical (includingbuccal, sublingual or transdermal), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous or intradermal) methods. Suchformulations can be prepared using all processes known in thepharmaceutical art by, for example, combining the active ingredient withthe excipient(s) or adjuvant(s).

Pharmaceutical formulations adapted for oral administration can beadministered as separate units, such as, for example, capsules ortablets; powders or granules; solutions or suspensions in aqueous ornon-aqueous liquids; edible foams or foam foods; or oil-in-water liquidemulsions or water-in-oil liquid emulsions.

Thus, for example, in the case of oral administration in the form of atablet or capsule, the active-ingredient component can be combined withan oral, non-toxic and pharmaceutically acceptable inert excipient, suchas, for example, ethanol, glycerol, water and the like. Powders areprepared by comminuting the compound to a suitable fine size and mixingit with a pharmaceutical excipient comminuted in a similar manner, suchas, for example, an edible carbohydrate, such as, for example, starch ormannitol. A flavour, preservative, dispersant and dye may likewise bepresent.

Capsules are produced by preparing a powder mixture as described aboveand filling shaped gelatine shells therewith. Glidants and lubricants,such as, for example, highly disperse silicic acid, talc, magnesiumstearate, calcium stearate or polyethylene glycol in solid form, can beadded to the powder mixture before the filling operation. A disintegrantor solubiliser, such as, for example, agar-agar, calcium carbonate orsodium carbonate, can likewise be added in order to improve theavailability of the medicament after the capsule has been taken.

In addition, if desired or necessary, suitable binders, lubricants anddisintegrants as well as dyes can likewise be incorporated into themixture. Suitable binders include starch, gelatine, natural sugars, suchas, for example, glucose or β-lactose, sweeteners made from maize,natural and synthetic rubber, such as, for example, acacia, tragacanthor sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes,and the like. The lubricants used in these dosage forms include sodiumoleate, sodium stearate, magnesium stearate, sodium benzoate, sodiumacetate, sodium chloride and the like. The disintegrants include,without being restricted thereto, starch, methylcellulose, agar,bentonite, xanthan gum and the like. The tablets are formulated by, forexample, preparing a powder mixture, granulating or dry-pressing themixture, adding a lubricant and a disintegrant and pressing the entiremixture to give tablets. A powder mixture is prepared by mixing thecompound comminuted in a suitable manner with a diluent or a base, asdescribed above, and optionally with a binder, such as, for example,carboxymethylcellulose, an alginate, gelatine or polyvinylpyrrolidone, adissolution retardant, such as, for example, paraffin, an absorptionaccelerator, such as, for example, a quaternary salt, and/or anabsorbent, such as, for example, bentonite, kaolin or dicalciumphosphate. The powder mixture can be granulated by wetting it with abinder, such as, for example, syrup, starch paste, acadia mucilage orsolutions of cellulose or polymer materials and pressing it through asieve. As an alternative to granulation, the powder mixture can be runthrough a tableting machine, giving lumps of non-uniform shape, whichare broken up to form granules. The granules can be lubricated byaddition of stearic acid, a stearate salt, talc or mineral oil in orderto prevent sticking to the tablet casting moulds. The lubricated mixtureis then pressed to give tablets. The compounds according to theinvention can also be combined with a free-flowing inert excipient andthen pressed directly to give tablets without carrying out thegranulation or dry-pressing steps. A transparent or opaque protectivelayer consisting of a shellac sealing layer, a layer of sugar or polymermaterial and a gloss layer of wax may be present. Dyes can be added tothese coatings in order to be able to differentiate between differentdosage units.

Oral liquids, such as, for example, solution, syrups and elixirs, can beprepared in the form of dosage units so that a given quantity contains apre-specified amount of the compound. Syrups can be prepared bydissolving the compound in an aqueous solution with a suitable flavour,while elixirs are prepared using a non-toxic alcoholic vehicle.Suspensions can be formulated by dispersion of the compound in anon-toxic vehicle. Solubilisers and emulsifiers, such as, for example,ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers,preservatives, flavour additives, such as, for example, peppermint oilor natural sweeteners or saccharin, or other artificial sweeteners andthe like, can likewise be added.

The dosage unit formulations for oral administration can, if desired, beencapsulated in microcapsules. The formulation can also be prepared insuch a way that the release is extended or retarded, such as, forexample, by coating or embedding of particulate material in polymers,wax and the like.

The conjugates according to the invention can also be administered inthe form of liposome delivery systems, such as, for example, smallunilamellar vesicles, large unilamellar vesicles and multilamellarvesicles. Liposomes can be formed from various phospholipids, such as,for example, cholesterol, stearylamine or phosphatidylcholines.

The conjugates according to the invention can also be delivered usingmonoclonal antibodies as individual carriers to which the conjugates arecoupled. The conjugates can also be coupled to soluble polymers astargeted medicament carriers. Such polymers may encompasspolyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamidophenol, polyhydroxyethylaspartamidophenolor polyethylene oxide polylysine, substituted by palmitoyl radicals. Thecompounds may furthermore be coupled to a class of biodegradablepolymers which are suitable for achieving controlled release of amedicament, for example polylactic acid, poly-ε-caprolactone,polyhydroxybutyric acid, polyorthoesters, polyacetals,polydihydroxypyrans, polycyanoacrylates and crosslinked or amphipathicblock copolymers of hydrogels.

Pharmaceutical formulations adapted for transdermal administration canbe administered as independent plasters for extended, close contact withthe epidermis of the recipient. Thus, for example, the active ingredientcan be delivered from the plaster by iontophoresis, as described ingeneral terms in Pharmaceutical Research, 3(6), 318 (1986).

Pharmaceutical compounds adapted for topical administration can beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils.

Pharmaceutical formulations adapted for rectal administration can beadministered in the form of suppositories or enemas.

Pharmaceutical formulations adapted for nasal administration in whichthe carrier substance is a solid comprise a coarse powder having aparticle size, for example, in the range 20-500 microns, which isadministered in the manner in which snuff is taken, i.e. by rapidinhalation via the nasal passages from a container containing the powderheld close to the nose. Suitable formulations for administration asnasal spray or nose drops with a liquid as carrier substance encompassactive-ingredient solutions in water or oil.

Pharmaceutical formulations adapted for administration by inhalationencompass finely particulate dusts or mists, which can be generated byvarious types of pressurised dispensers with aerosols, nebulisers orinsufflators.

Pharmaceutical formulations adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions comprisingantioxidants, buffers, bacteriostatics and solutes, by means of whichthe formulation is rendered isotonic with the blood of the recipient tobe treated; and aqueous and non-aqueous sterile suspensions, which maycomprise suspension media and thickeners. The formulations can beadministered in single-dose or multidose containers, for example sealedampoules and vials, and stored in freeze-dried (lyophilised) state, sothat only the addition of the sterile carrier liquid, for example waterfor injection purposes, immediately before use is necessary. Injectionsolutions and suspensions prepared in accordance with the recipe can beprepared from sterile powders, granules and tablets.

The conjugates according to the invention are preferably administeredparenterally.

It goes without saying that, in addition to the above particularlymentioned constituents, the formulations may also comprise other agentsusual in the art with respect to the particular type of formulation;thus, for example, formulations which are suitable for oraladministration may comprise flavours.

A therapeutically effective amount of the conjugate according to theinvention depends on a number of factors, including the type of coupledactive compound, the age and weight of the patient, the precisecondition that requires treatment, and its severity, the nature of theformulation and the method of administration.

The present invention also relates to a kit for the preparation of apharmaceutical composition, in particular an image-enhancing ortherapeutic composition, at least comprising a conjugate according tothe invention. The conjugate according to the invention can be in thekit in dissolved form in a solvent (for example an aqueous buffer) orpreferably in the form of the lyophilisate.

It has been found that the conjugates according to the invention havealready accumulated specifically, i.e. exclusively or virtuallyexclusively, in the kidney a short time after application. In the caseof the preferred intravenous administration of the conjugates accordingto the invention, concentration in the kidney is observed after only 5minutes. After one hour, more than 30%, preferably more than 50%,particularly preferably more than 70%, very particularly preferably morethan 80%, of the injected dose is located in the kidney (% data based onmeasurement of the radioactivity).

In organ distribution studies with radiolabelled conjugates according tothe invention (for example PET measurements or other non-invasiveimaging), the conjugates according to the invention typically exhibit atleast a two-fold, preferably at least a five-fold, particularlypreferably at least a ten-fold concentration in the kidney in relationto the remainder of the body (blood, heart, lung, spleen, liver, muscle,brain) one hour after application. This means that the signal, whichcorrelates directly with the amount of radiolabelled compound, in thekidney is at least twice as strong as the sum of the signals obtainedfrom blood, heart, lung, spleen, liver, muscle and brain together.

In accordance with the invention, targeting of the kidney means theachievement of increased uptake of the applied substance in the kidneyin relation to the remainder of the body. In the case of targeting ofthe kidney with the conjugate according to the invention, at least atwo-fold, preferably at least a five-fold, particularly preferably atleast a ten-fold concentration is preferably achieved in the kidney inrelation to the remainder of the body (blood, heart, lung, spleen,liver, muscle, brain) by administration of a conjugate according to theinvention. These values are determined by means of organ distributionstudies with radiolabelled conjugates according to the invention (forexample PET measurements or other non-invasive imaging).

The concentration in the kidney typically takes place after 30 minutesto 8 hours, depending on the type of application.

FIGURES

FIG. 1 shows the influence of the chain length on the release of activecompound for the structures MAG3-KKEEEKKEEEKKEEEK andMAG3-KKEEEKKEEEKKEEEKKEEEKKEEEKKEEE (N-terminal linking of the activecompound —FIG. 1, top) and KKEEEKKEEEKKEEE-y andKKEEEKKEEEKKEEEKKEEEKKEEEKKEEE-y (C-terminal linking of the activecompound —FIG. 1, bottom).

FIG. 2 shows the influence of the chain length on the release of activecompound for the structure y-KKEEEKKEEEKKEEEK (N-terminal linking of theactive compound —FIG. 2, bottom) and the structures KKEEEKKEEEKKEEE-yand KKEEEKKEEEKKEEEKKEEEKKEEEKKEEE-y (C-terminal linking of the activecompound —FIG. 2, top).

FIG. 3 compares the organ distribution of the 125iodine-labelledconjugate y(KKEEE)₃K depending on the administration route.

FIG. 4 shows the scintigraphic distribution of the diacetylcaffeic acid(KKEEE)₃K active compound conjugate bonded at the N terminal afterintravenous administration in an NMRI mouse.

FIG. 5 shows the scintigraphic distribution of the diconjugated moleculeyKKK(DCA)EEEKKEEEKKK(DCA)EEEK (CDA=diacetylcaffeic acid) afterintravenous administration in an NMRI mouse.

FIG. 6 shows the scintigraphic distribution of ¹²⁵I-y(KKKε(lipoicacid)EEE)₃K after intravenous administration in an NMRI mouse.

Even without further comments, it is assumed that a person skilled inthe art will be able to utilise the above description in the broadestscope. The preferred embodiments and examples should therefore merely beregarded as descriptive disclosure which is absolutely not limiting inany way.

EXAMPLES 1. Material Syntheses

1.1. Synthesis of the Peptides Containing Acidic and Basic Side Groups

Solid-Phase Peptide Synthesis

The peptides are prepared on an ABI 433A fully automatic peptidesynthesiser from Applied Biosystems GmbH (Carlsbad, Calif., USA) inaccordance with the Fmoc/tBu strategy using Tentagel S RAM resin (degreeof loading: 0.24 mmol/g; Rapp Polymere, Tübingen, Germany) as polymericsupport. Fmoc-amino acids (Fmoc-AA-OH; Novabiochem, Merck KGaA,Darmstadt, Germany) containing acid-labile side-chain protecting groups(for example Arg(Pbf), Asn(Trt), Asp(tBu), Cys(Trt), Gln(Trt), Glu(tBu),His(Trt), Lys(Boc), Ser(tBu), Thr(tBu), Tyr(tBu)) are used as startingmaterials. The synthesis cycle consists of a) cleaving-off of the Fmocprotecting group using 20% piperidine in NMP, b) washing steps with NMP,c) coupling: Fmoc-AA-OH/HBTU/DIPEA/peptide resin 10/10/20/1, 8 min, d)washing steps with NMP.

The effectiveness of the cleaving-off of Fmoc are monitored by means ofautomatic conductivity measurements. The peptides are cleaved off fromthe resin using TFA/H₂O/triisopropylsilane (95:2.5:2.5) (2 h at roomtemperature), precipitated out in cold methyl tert-butyl ether,separated by means of centrifugation (4000 rpm, 5 min), dried in vacuoand lyophilised from MeCN/H₂O (1:1).

Purification and Characterisation of Peptides

The purification of the peptide cleaved off from the resin is carriedout by means of semipreparative HPLC using an LaPrep unit (VWR GmbH,Darmstadt, Germany). The column used is a Waters XBridge BEH130 PREP C18(5 μm, 19×150 mm) column (flow rates: 9-20 ml/min; solvent: 0.1% of TFAin water to 0.1% of TFA in acetonitrile). The separation is carried outusing a gradient from water to acetonitrile which is matched to thephysicochemical properties of the corresponding peptides. The purifiedpeptide is obtained after lyophilisation.

For characterisation, the peptides prepared are analysed by means ofanalytical HPLC (Agilent 1100) and HPLC-MS (Exactive, Thermo FisherScientific). The HPLC analysis under standard conditions is carried outon the basis of a linear gradient from 0.1% of TFA in water to 0.1% ofTFA in acetonitrile in 5 min (conditions: ChromolithR Performance RP-18ecolumn, 100×3 mm; flow rate: 2 ml/min, wavelength=214 nm). For the massspectrometry, an Agilent 1200 serves as HPLC system (conditions:Hypersil Gold C18 column, 0.21×200 mm, gradient: from 0.05% of TFA inwater to 0.05% of TFA in acetonitrile in 30 min, flow rate: 200 μl/min,column oven: 60° C., wavelength=214 nm).

Radioactive Iodination of Peptides

The labelling is carried out using a 1 mM stock solution of the peptideto be labelled in water (DMSO may have to be added for bettersolubility). Tyrosine-containing peptides are labelled with iodine-123,iodine-125 or iodine-131 by means of the chloramine-T method(Perkin-Elmer, Waltham, Mass., USA). To this end, 20 μl of phosphatebuffer (0.25 M, pH 7.4) are added to 10 μl of the stock solution, andthe desired amount of radioactive iodine is added. For the labelling, 5μl of chloramine-T (2 mg/ml of H₂O) are added. The reaction is carriedout for 30 seconds and is subsequently terminated using 10 μl of asaturated methionine solution. In order to separate off free iodine andby-products, the reaction mixture is purified by means ofsemipreparative HPLC (Chromolith RP-18e, 100×4.6 mm). The separation iscarried out using a linear gradient from 0.1% of TFA in water to 0.1% ofTFA in acetonitrile in 10 minutes (flow rate: 2 ml/min, UV absorption at214 nm, γ detection). The solvent is subsequently removed in a rotaryevaporator, and the labelled peptide is taken up in the desired buffer.

1.2. Synthesis of the ε-L-Polylysine Conjugates

ε-L-Polylysine-DOTA:

DOTA 2,6-Difluorophenyl Ester:

From DOTA and 2,6-difluorophenol with DCC (Mier et al. BioconjugateChem. 2005, 16, 237) ε-L-Polylysine, average molar mass about 4000(principally consisting of 29-34 lysine units), is purchased as 25%aqueous solution from Chisso Corp. (Japan) and lyophilised. ε-Polylysine(30 mg) is dissolved in water (200 μl), and a solution of DOTA2,6-difluorophenyl ester (100 mg) in methanol (1 ml) is added, and 100μl of N,N-diisopropylethylamine are added, and the mixture is stirred atRT for 2 days. DOTA 2,6-difluorophenyl ester (100 mg) is then againadded, and the mixture is stirred overnight at RT. The mixture is thendiluted with water and purified preparatively by HPLC. Clean fractionsare lyophilised together. DOTA-ε-polylysine (98 mg) is obtained ascolourless solid substance. The number of DOTA units per molecule ofε-polylysine is determined by loading with Gd and by MS as being about10 DOTA units per molecule of ε-polylysine; i.e. about 30% of the aminogroups of the ε-polylysine have reacted.

ε-L-Polylysine-DTPA

75 mg of ε-L-polylysine and 310 mg of DTPA difluorophenyl estertetra-t-butyl ester are dissolved in 4 ml of methanol and stirred at RTfor 20 h. The reaction solution is evaporated, and 4 ml of TFA+100 μl ofwater are added to the residue and left to stand for 20 h. The productis precipitated using diethyl ether. Purification by HPLC andlyophilisation gives 150 mg as colourless solid.

1.3. Preparation of Lipoic Acid-y(KKEEE)₃K

The peptide y(KKEEE)₃K is prepared in a peptide synthesiser as describedunder 1.1 by means of solid-phase synthesis of the Fmoc/tBu strategyusing the amino acids Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH andFmocTyr(tBu)-OH (Novabiochem, Merck KGaA, Darmstadt, Germany). Thepeptide is initially not cleaved off from the resin, but insteadsuspended in NMP after the final Fmoc deprotection (1 ml of NMP are usedper 100 mg of peptide resin). (RS)-lipoic acid (Merck KGaA, Darmstadt,Germany; in the meantime 4 equivalents based on the resin loading) isdissolved in NMP (1 ml per 100 mg), HBTU (4 eq.) is added, and themixture is stirred at room temperature for about 10 min. The reactionmixture is added to the peptide resin, DIPEA (10 eq.) is added, and themixture is shaken at room temperature for about 4 h. The resin is washed5× with NMP and 5× with DCM and dried in vacuo for about 4 h. The lipoicacid/peptide conjugate is cleaved off from the resin usingTFA/thioanisole/EDT/anisole (90/5/3/2) at room temperature for about 1h, precipitated out in cold methyl tert-butyl ether, separated by meansof centrifugation (4000 rpm, 5 min), dried in vacuo, lyophilised fromMeCN/H₂O (1:1) and purified as described under 1.1 Purification andcharacterisation of the peptides.

Conjugates with other active compounds can also be prepared analogously.

1.4 Preparation of yKKK(Diacetylcaffeic Acid)(EEEKK)₂K(DiacetylcaffeicAcid)EEEK

For the peptic conjugation of diacetylcaffeic acid onto a lysine sidechain, the amino acid Fmoc-Lys(Mmt)-OH is incorporated into the sequenceof the peptide backbone. Before the cleaving-off, dichloromethane(DCM)/triisopropylsilane/TFA (94:5:1) is added to the peptide resinprepared under 1.1 for 3 min, and the mixture is washed 5× with DCM.This operation is repeated 3×. For coupling to the orthogonallydeprotected side chain of lysine, 4 eq of diacetylcaffeic acid aredissolved in NMP, 4 eq of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC), 4 eq of ethyl cyano(hydroxyimino)acetate (Oxyma Pure) and 10 eqof diisopropylethylamine (DIPEA) are added, the mixture is stirred atroom temperature for about 10 min and subsequently added to the peptideresin. The reaction mixture is shaken at room temperature for about 1 h,washed 5× with NMP and 5× with DCM and dried in vacuo. Thefunctionalised peptide is cleaved off from the resin and purified asdescribed under 1.1.

Conjugates with other active compounds can also be prepared analogously.

1.5 Preparation of y(KKKε(Lipoic Acid)EEE)₃K

1.5.1 Synthesis of the Fmoc-Lysine(ε-Lipoic Acid)-OH Building Block

N-Hydroxysuccinimide (1.15 g, 10 mmol), α-lipoic acid (2.02 g, 9.8 mmol)and (1.92 g, 10 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDAC) are dissolved in 50 ml of DMF and stirred at room temperature forabout 4 h. 60 ml of ethyl acetate are then added to the batch. Theorganic phase is washed three times with 60 ml of distilled water, threetimes with 60 ml of saturated sodium hydrogencarbonate solution and oncewith saturated sodium chloride solution. The ethyl acetate phase isdried over Na₂SO₄, filtered and evaporated to dryness.

Yield: 2.23 g (73.5%)

Fmoc-Lys-OH (2.65 g, 7.2 mmol) is suspended in 110 ml of HEPES buffer(pH=7.4), and (2.14 g, 7.05 mmol) of lipoic acid active ester (dissolvedin 130 ml of acetone) are added, and the mixture is stirred at roomtemperature. After a reaction time of about 3 h, the solution isadjusted to pH 7 by means of 0.1 N NaOH solution and stirred at roomtemperature for about 20 h. The batch is then brought to pH 9 using 0.1N NaOH and washed twice with about 30 ml of ethyl acetate, subsequentlyadjusted to pH 3 using 1 N HCl and extracted three times with about 40ml of ethyl acetate. The combined org. phases are washed with saturatedsodium chloride solution, dried over Na2SO4, filtered and evaporated todryness.

Weight of crude product: 4.14 g (103. 25%)

The purification of the crude product is carried out by flashchromatography (stationary phase: silica gel 60, particle size: 15-40μm, pre-packed by Götec-Labortechnik GmbH, mobile phase: chloroform,methanol (comprising 0.1% of HOAc), flow rate: 60 ml/min, loading: about2 g, gradient: from 100% to 75% of chloroform in 18 min). The productfractions (Rt=9.1 min) are combined and evaporated to dryness.

Product weight: 3.18 g (77%)

1.5.2 Solid-Phase Peptide Synthesis

Peptides are prepared using a synthesiser from Applied Biosystems GmbH(Carlsbad, Calif., USA), model 433A, using the Fmoc/tBu strategy. Thereactive side chains of the amino acids are protected as follows:Lys(Boc), Glu(tBu) and Tyr(tBu). Rink amide resin from Rapp-PolymereGmbH (degree of loading: 0.24 mmol/g) serves as solid phase. Thecorresponding amino acids, the Fmoc-lysine(ε-lipoic acid)-OH buildingblock and HBTU are employed in 4-fold excess. The solvent used is NMP,and piperidine (20% in NMP) is used for the respective Fmoc cleavingoff.

The protected peptide is cleaved off from the resin usingTFA:thioanisole:anisole=90:8:2 (1 ml per 100 mg) (1-2 h), precipitatedout in MTBE, centrifuged and dried.

1.5.3 Radioactive Iodination of Peptides

The tyrosine-containing peptides are labelled with ¹²⁵iodine by means ofthe chloramine-T method. For the labelling, a 1 mM stock solution inwater is used. If necessary, DMSO is added for better solubility. Tothis end, 20 μl of phosphate buffer (0.25 M, pH 7.4) are added to 10 μlof the stock solution, and the desired amount of radioactive iodine isadded. The labelling is carried out using 5 μl of chloramine-T (2 mg/mlof H₂O). The reaction is carried out for 30 seconds and is subsequentlyterminated using 10 μl of a saturated methionine solution.

After the labelling, the peptide is purified by means ofsemi-preparative HPLC in order to remove the excess free iodine andother by-products. 100 μl of the 0.1 mM stock solution are in each caseused for the injection. Before the injection, the radioactivity isrecorded by means of a Geiger counter.

Conjugates with other active compounds can also be prepared analogously.

2. Use Examples

2.1. Organ Distribution Studies

In order to determine the pharmacokinetics, the radioactively labelledmolecules from Example 1.1 to be investigated are injected into femaleNMRI mice via the tail vein (about 100 μl per animal). The animals (n=3per time point) are subsequently sacrificed at the corresponding timepoints, dissected, and the distribution of the radioactivity in theisolated organs (liver, kidney, lung, spleen, intestine, brain, heart,blood, . . . ) is quantified by y counter (Berthold LB951G). Theradioactivity measured per gram of organ/tissue based on the injecteddose (ID) is determined and quoted as % of ID/g.

The influence of the chain length on the release of active compound isinvestigated. The structures MAG3-KKEEEKKEEEKKEEEK,MAG3-KKEEEKKEEEKKEEEKKEEEKKEEEKKEEE and y-KKEEEKKEEEKKEEEK (N-terminallinking of the active compound —FIG. 1, top and FIG. 2 bottom) and thestructures KKEEEKKEEEKKEEE-y and KKEEEKKEEEKKEEEKKEEEKKEEEKKEEE-y(C-terminal linking of the active compound —FIG. 1, bottom and FIG. 2,top) are investigated y here stands for tyrosine; MAG3 stands for apeptide fragment which complexes ^(99m)Tc.

The result is depicted in FIGS. 1 and 2 (ID/g here stands for “injecteddose per gram of tissue): the release of radiolabelled tyrosine (as“active compound”) is strongly influenced on the one hand via the chainlength and on the other hand via the linking site of the “activecompound” (C or N terminal). Basically, longer peptides result indelayed release. In addition, the release of tracers linked at the Cterminal (iodotyrosine or also MAG3 with ^(99m)Tc) proceedssignificantly more quickly than in the case of N-terminal linking. Therate-determining step in the peptide degradation is apparentlyinfluenced, in particular, by carboxypeptidases, which break down thepeptide starting from the C terminal.

The release kinetics of an active compound can be intentionally adjustedthrough the molecular structure of the peptide and the linking site ofthe active compound (C or N terminal).

2.2. Administration Route

In further experiments, the administration route is investigated. Tothis end, nine NMRI mice are divided into three groups. All animalsreceive 10 mg/kg of body weight of a conjugate of D-tyrosine bonded to(KKEEE)₃K at the N terminal. Part of the conjugate is labelled with 131iodine on the D-tyrosine by means of the chloramine-T method. Thelabelled conjugate is administered intravenously to group 1,subcutaneously to group 2 and intraperitoneally to group 3. Theconjugate here is dissolved in 100 μl of PBS buffer. SPECT scans ofanimals from the respective group are then carried out at various times(40, 60, 120 and 240 minutes). The results of this experimental seriesare depicted in FIG. 3. Besides the intravenous administration ofpeptides/proteins described in the literature for transport of activecompound into the kidneys, subcutaneous and intraperitonealadministration of the peptides or peptide/active compound conjugatesaccording to the invention can also successfully address the kidneys.

2.3. Scintigraphic Distribution of Diacetylcaffeic Acid Conjugates

In further experiments, the potential active compound diacetylcaffeicacid (DCA) is bonded both at the N terminal and also multiply to lysineside chains of the peptide backbone. The preparation of the N-terminalconjugate with y(KKEEE)₃K is carried out analogously as described under1.3.; the preparation of the diconjugated molecule (structure:yKKK(DCA)EEEKKEEEKKK(DCA)EEEK) is carried out analogously as describedunder 1.4. The peptide/active compound conjugates obtained in this wayare investigated for their kidney selectivity after labelling by meansof iodine-125 and intravenous administration in the animal model mouse.

Result (see FIGS. 4 and 5): the peptide/active compound conjugatesprepared retain their high kidney specificity both after N-terminalbonding of diacetylcaffeic acid (FIG. 4) and also in the case of doublebonding of diacetylcaffeic acid to different side chains of lysine ofthe peptide backbone (FIG. 5).

2.4. Protection of the Kidney

In a preclinical study, BALB/c mice are treated with doxorubicin. Eachexperimental animal receives a dose of 11 mg/kg of body weight. Acontrol group is merely administered an isotonic saline solution. Theanimals treated with doxorubicin are divided into various groups. GroupB receives an injection of free lipoic acid in the form of its potassiumsalt in addition to the doxorubicin injected. In the case of group C,the lipoic acid/peptide conjugate LA-(KKEEE)₃ (lipoic acid on(Lys-Lys-Glu-Glu-Glu)₃ N terminal) is administered by injection insteadof free lipoic acid.

Doxorubicin + lipoic acid Control Doxorubicin conjugate Doxorubicin +lipoic acid Number of 6 6 6 per dose; in the case of 6 per dose; in thecase of animals 3 doses = 18 animals 3 doses = 18 animals Treatment 0.9%salt Doxorubicin hydrochloride Doxorubicin hydrochloride Doxorubicinhydrochloride solution diluted to 11 mg/kg diluted to 11 mg/kg dilutedto 11 mg/kg of body weight in of body weight in of body weight in 0.9%salt solution 0.9% salt solution 0.9% salt solution2.5 Scintigraphic Distribution of Lipoic Acid Conjugates in Accordancewith Example 1.5

In further experiments, the potential active compound lipoic acid isbonded via the lysine side chains of the peptide backbone. Thepreparation of the conjugate y(KKKε(lipoic acid)EEE)₃K is carried out asdescribed in Example 1.5. The peptide/active compound conjugate obtainedin this way is investigated for its kidney selectivity after labellingby means of iodine-125 and intravenous administration in the animalmodel mouse.

Result (see FIG. 6): the peptide/active compound conjugate prepared hashigh kidney specificity.

The invention claimed is:
 1. A conjugate containing at least onekidney-selective carrier molecule and at least one active, wherein theat least one kidney-selective carrier molecule is an oligopeptidecomprising multiple monomeric peptides comprising more than 50% (basedon the number of amino acid units) of sequence sections selected fromthe group consisting of KKEE (SEQ ID NO: 78), KKEEE (SEQ ID NO: 76),RREEE (SEQ ID NO: 77), KKKEEE (SEQ ID NO: 79) and KKKEE (SEQ ID NO: 80),wherein (i) the peptide or oligopeptide overall has a chain length of 15to 100 amino acid units; (ii) the peptide or oligopeptide comprises atleast 50% (based on the number of amino acid units) of amino acids K andE, or R and E, respectively; and (iii) the oligopeptide comprises atleast 3 consecutive sequence sections.
 2. The conjugate of claim 1,wherein the oligopeptide contains 3 to 5 successive sequence sections.3. The conjugate of claim 1, wherein the oligopeptide is selected fromthe group consisting of (RREEE)₃R (SEQ ID NO: 81), (KKEE)₅K (SEQ ID NO:82), (KKKEE)₃K (SEQ ID NO: 83), (KKKEEE)₃K (SEQ ID NO: 84) and (KKEEE)₃K(SEQ ID NO: 85).
 4. The conjugate of claim 1, wherein the at least oneactive compound is an antioxidant and/or an apoptosis inhibitor.
 5. Apharmaceutical composition comprising at least one conjugate of claim 1.6. The pharmaceutical composition of claim 5, further comprising apharmaceutically acceptable excipient.
 7. The conjugate of claim 4,wherein the at least one active compound is selected from the groupconsisting of lipoic acid, resveratrol, caffeic acid, luteolin,quercetin, rutin, cyanidin, xanthohumol, ascorbic acid, nicotinic acid,amifostin, alliin, thiols, tocopherols, carotinoids, butylhydroxytoluene(BHT), pifithrin-μ, pifithrin-α, MDL 28170 and NS3694 and combinationsthereof.
 8. A method for the protection of the kidney againstnephrotoxic active compounds, comprising administering to a patient inneed thereof an effective amount of a pharmaceutical composition ofclaim 7.